Trizol-1-ol analogs anti-retroviral latency drugs

ABSTRACT

In one aspect, the invention relates to triazol-1-ol compounds, analogs thereof, compositions comprising same, and methods of using same, alone or in combination with other agents, to reactivate latent retroviruses, and more particularly to reactivate latent HIV-1. Such compounds, compositions, and methods can be used, for example, in connection with diagnosing and/or treating a retrovirus, and more specifically HIV-1. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application No.61/835,297, filed on Jun. 14, 2013, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. R01 AI087508-01A1 awarded by the National Institutes of Health. The UnitedStates government has certain rights in the invention.

BACKGROUND

The advent of highly active anti-retroviral therapy (HAART), whichinvolves the use of three or more antiretroviral drugs, has led to asignificant improvement in the care and survival of patients infect withHIV-1. In patients not infected with resistant strains of the virus,HAART typically results in a dramatic decrease in viral load often fromlevels of 10,000-100,000 RNA copies/mL of plasma to less than 50copies/mL.

Given the dramatic effects of HAART, it was proposed that completeelimination of the virus might be possible within 2 to 3 years. However,even after long-term suppression of viral replication with HAART, thevirus rapidly rebounds after therapy is discontinued. A key contributorto viral rebound appears to be a reservoir of latently infected cells,including CD4 memory T cells. The half-life of the latently infectedpopulation is quite long, and it is estimated that it would take over 60years of HAART to eliminate this population. Therefore, life-long HAARTwould be required to control infection in patients

Retroviruses, including HIV-1, are RNA viruses that replicate through aDNA intermediate and integrate very efficiently into the genome of aninfected cell, forming a provirus. Once the provirus is formed, it ismaintained in the genome of the infected cell and transferred todaughter cells in the same fashion as any other genetic element withinthe cellular genome. Thus, the virus has the potential to persist if itinfects long-lived cells such as memory T cells. It has been known since1986 that HIV-1 can establish a latent infection in culture. It wasfound that a human T cell line infected with replication-competent viruscould develop a latent infection in which the provirus was dormant butcould be reactivated upon stimulation. Since then it has beenestablished that a number of cytokines can reactivate latent proviruses.

The role that latency is playing in preventing clearance of the virusinfection has become evident in recent years. Patients that had beensuccessfully treated with HAART in which viral RNA was maintained atlevels below 50 copies/mL in the plasma for years, experienced rapidvirus rebound upon withdrawal of therapy. Moreover, it was found thatafter T cell activation, virus could be isolated from CD4 T cells takenfrom these patients making it clear that to eradicate the virus it willbe necessary to eliminate the latently infected cells.

There have been attempts to flush the latent virus from infectedindividuals by nonspecific activation of T cells to “turn on” latentproviruses. As part of this approach, the patients remain on HAART toprevent new infections, and the infected cells from which the latentproviruses are activated should die due to cytotoxic effects of viralexpression and/or because of targeting by the immune system which canrecognize the cells once they begin to express the viral proteins.Agents that can dissociate latent virus activation from global T cellactivation, however, are extremely rare. Thus, there remains a need fornew drugs capable of selectively activating latent viruses.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates tomethods of activating a latent retrovirus in a subject, the methodcomprising the step of administering to the subject an effective amountof a compound represented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring.

Also disclosed are methods for the manufacture of a medicament fortreatment of a retrovirus in a subject, the method comprising the stepof combining an effective amount of a compound represented by a formula:

wherein n is 0 or 1, wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), or1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; wherein R³ is selectedfrom H and C1-C4 alkyl, or R² and R³ are covalently bonded and, togetherwith the intermediate atoms, comprise an optionally substituted fusedsix-membered aryl or heteroaryl ring; with a pharmaceutically acceptablecarrier or diluent.

Also disclosed are kits comprising at least one compound represented bya formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring; and one or more of

-   -   a) at least one agent known to treat a retrovirus;    -   b) instructions for detecting a retrovirus; and    -   c) instructions for treating a retrovirus.

Also disclosed are pharmaceutical compositions comprising a compoundrepresented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; wherein R³ is selectedfrom H and C1-C4 alkyl, or R² and R³ are covalently bonded and, togetherwith the intermediate atoms, comprise an optionally substituted fusedsix-membered aryl or heteroaryl ring; and a pharmaceutically acceptablecarrier or diluent.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1A shows the results of a screen of the Ireland Natural ProductCollection, a set of natural products isolated from marine invertebrateanimals and microorganisms. Plate II well C07 was identified as apositive result and was taken on for further analysis.

FIG. 1B shows that Plate II well C07 activates latent HIV-1 as indicatedby an increase in fluorescence compared to the control.

FIG. 1C shows the % of positive cells and the % of reactivation comparedto the positive control for 20 fractions of C07. The fractions wereseparated using HPLC with a gradient of 10-100% in acetonitrile with0.1% formic acid. The activity of CD3/CD28 is shown as a positivecontrol. The ability of C07 (MNP-II C07) to activate latent HIV-1 isalso displayed.

FIG. 1D shows that the compound responsible for the activity of C07 islocated in fraction MS. The major component of this fraction is1-hydroxybenzotriazole (HOBt).

FIG. 1E shows the structure of HOBt.

FIG. 1F shows that HOBt reactivates HIV-1 latency in a dose dependentmanner.

FIG. 1G shows that HOBt is able to reactive HIV-1 latency with slowerkinetics compared to anti-CD3/CD28.

FIG. 2A shows the ability of HOBt and HOBt analogs to activate latentHIV-1. In addition to HOBt, HOAt also activates HIV-1 latent cells.

FIG. 2B shows that both HOBt and HOAt activate latent HIV-1.

FIG. 2C shows that HOBt and HOAt reactivate HIV-1 latency in a dosedependent manner.

FIG. 2D shows the ability of a panel of 7 derivatives of HOBt toactivate latent HIV-1. HOAt demonstrates increased activity compared toHOBt. Two additional analogs, HATU and HO-DhBt, also illustrate anincreased ability to induce viral reactivation in a dose dependentmanner.

FIG. 3 shows that HOBt and HOAt synergize with several γc-cytokines toreactivate HIV-1 latency. HOBt and HOAt increase viral reactivation inthe presence of IL-2, IL-7 and IL-5. IL-4 did not induce viralreactivation by itself; however, combining IL-4 with either HOBt or HOAtdid induce significant viral reactivation. IL-9 and IL-21 show noeffect.

FIG. 4A shows that HOAt induces viral reactivation in a STAT5 dependentmanner. Inhibition of either NFAT or NF-κB did not have an effect onviral reactivation by HOAt. Inhibition of JAK selectively blocked viralreactivation by HOAt but not by αCD3/αCD28. While the STAT3 inhibitordid not have a significant effect, the STAT5 inhibitor was able tocompletely prevent activation of latent HIV-1 by HOAt.

FIG. 4B shows that HOAt alone is able to induce an increase in STAT5phosphorylation over untreated cells. While cells incubated with IL-2alone increase the levels of STAT5 phosphorylation at 30 min, at 24 hrsthe level has decreased to basal levels. Incubation of cultured T_(CM)cells with a combination of IL-2 and HOAt lead to a drastic increase inSTAT5 phosphorylation.

FIG. 5A shows that HOAt increases the nuclear localization of STAT5.pSTAT5 and STAT5 levels increased in the nucleus of the cells treatedwith HOAt at the time points indicated. Histone H3 and alpha Tubulinwere used as controls for purity of the fractionation.

FIG. 5B shows that IL-2 alone or in the presence of HOAt increasesphosphorylation of JAK1 and JAK3.

FIG. 5C shows that HOAt increases the transcription of SOC3S, a targetgene for STAT5.

FIG. 6A shows that HOBt does not induce general T cell activation.

FIG. 6B shows that HOBt does not induce T cell differentiation measuredas lost CCR7 and CD27 expression.

FIG. 6C shows that HOBt only induces some degree of T cell proliferationat the higher concentration tested.

FIG. 61D shows that HOBt only induces an increase of 1.4 times of CD25levels at the maximum concentration used. This level is increased by 37times after TCR engagement with αCD3/αCD28.

FIG. 6E shows that HOAt slightly increases the levels of T cellactivation when compared with αCD3/αCD28.

FIG. 7 shows that HOAt increases viral reactivation mediated by PMA,ionomycin and SAHA. This indicates that HOAt and additional analogs canbe used to synergize the activity of other anti-latency drugs.

FIG. 8 shows representative data pertaining to the ability of HOAt toreactivate latent HIV-1 in cells isolated from aviremic HIV-1 patients.Briefly, cells for two patients suppressed with antiretroviral wereisolated and treated with either DMSO (Control), HOAt or the mitogenPHA. As it is shown, HOAt can induce regrowth of latent viruses in bothpatients in vitro above the treatment control (DMSO).

FIG. 9A shows representative data demonstrating that HOAt increases thelevels of pSTAT5 in the inputs. FIG. 9B shows representative dataillustrating the ladder pattern normally observed in ubiquitinilatedproteins. Without wishing to be bound by theory, this may suggest thatHOAt and derivatives may block ubiquitation and subsequent degradationof pSTAT5. Briefly, cells were treated with DMSO or HOAt for 30 min, andsubsequently treated with IL-2 for another 30 min. STAT5 wasimmunoprecipitated with antibodies specific for the phosphorylatedprotein and western blot was performed against pSTAT5.

FIG. 10 shows representative data pertaining to the ability of HOBt,HOAt, HODHBt and HATU to maintain pSTAT5 levels in a dose dependentmanner. Cells were treated with IL-2 and several derivatives of HOAt.Levels of pSTAT5 were measured 48 h later by flow cytometer.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examples andFigures included herein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon. Nothing herein is tobe construed as an admission that the present invention is not entitledto antedate such publication by virtue of prior invention. Further, thedates of publication provided herein may be different from the actualpublication dates, which can require independent confirmation.

A. DEFINITIONS

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asCHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or can not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” can be a vertebrate, such as amammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject ofthe herein disclosed methods can be a human, non-human primate, horse,pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In one aspect, the subject is a mammal. A patient refers to asubject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder, preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder, and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In various aspects, the term covers anytreatment of a subject, including a mammal (e.g., a human), andincludes: (i) preventing the disease from occurring in a subject thatcan be predisposed to the disease but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, i.e., arresting its development;or (iii) relieving the disease, i.e., causing regression of the disease.In one aspect, the subject is a mammal such as a primate, and, in afurther aspect, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse,rabbit, rat, guinea pig, fruit fly, etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aclinical interview and/or a physical examination by a person of skill,for example, a physician, and found to have a condition that can bediagnosed or treated by the compounds, compositions, or methodsdisclosed herein. For example, “diagnosed with a depression disorder”means having been subjected to a clinical interview and/or physicalexamination by a person of skill, for example, a physician, and found tohave a condition that can be diagnosed or treated by a compound orcomposition that can cure, alleviate, prevent, or otherwise treat adepression disorder.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, buccal administration, andparenteral administration, including injectable such as intravenousadministration, intra-arterial administration, intramuscularadministration, and subcutaneous administration. Administration can becontinuous or intermittent. In various aspects, a preparation can beadministered therapeutically; that is, administered to treat an existingdisease or condition. In further various aspects, a preparation can beadministered prophylactically; that is, administered for prevention of adisease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target, either directly; i.e., by interacting with the targetitself, or indirectly; i.e., by interacting with another molecule,co-factor, factor, or protein on which the activity of the target isdependent.

As used herein, the term “effective amount” refers to an amount that issufficient to achieve the desired result or to have an effect on anundesired condition. For example, a “therapeutically effective amount”refers to an amount that is sufficient to achieve the desiredtherapeutic result or to have an effect on undesired symptoms, but isgenerally insufficient to cause adverse side affects. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder, the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of a compound at levels lowerthan those required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration. Consequently, single dose compositions cancontain such amounts or submultiples thereof to make up the daily dose.The dosage can be adjusted by the individual physician in the event ofany contraindications. Dosage can vary, and can be administered in oneor more dose administrations daily, for one or several days. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. In further various aspects, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e, without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, and N-oxides of a parent compound.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in certain aspects, their recovery,purification, and use for one or more of the purposes disclosed herein.

By “virlon,” “viral particle,” or “retroviral particle” is meant asingle virus minimally composed of an RNA or DNA genome, Pol protein(for reverse transcription of the RNA genome following infection), Gagprotein (structural protein present in the nucleocapsid), and anenvelope protein. As used herein, the RNA genome of the retroviralparticle is usually a recombinant RNA genome, e.g., contains an RNAsequence exogenous to the native retroviral genome and/or is defectivein an andogenous retroviral sequence (e.g., is defective in pol, gag,and/or env, and, as used herein, is normally defective in all threegenes).

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labeled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, respectively. Compounds further comprise prodrugs thereof andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of this invention. Certainisotopically-labeled compounds of the present invention, for examplethose into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds of the present invention and prodrugsthereof can generally be prepared by carrying out the procedures below,by substituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent.

The compounds described in the invention can be present as a solvate. Insome cases, the solvent used to prepare the solvate is an aqueoussolution, and the solvate is then often referred to as a hydrate. Thecompounds can be present as a hydrate, which can be obtained, forexample, by crystallization from a solvent or from aqueous solution. Inthis connection, one, two, three or any arbitrary number of solvate orwater molecules can combine with the compounds according to theinvention to form solvates and hydrates. Unless stated to the contrary,the invention includes all such possible solvates.

The term “co-crystal” means a physical association of two or moremolecules which owe their stability through non-covalent interaction.One or more components of this molecular complex provide a stableframework in the crystalline lattice. In certain instances, the guestmolecules are incorporated in the crystalline lattice as anhydrates orsolvates, see e.g. “Crystal Engineering of the Composition ofPharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a NewPath to Improved Medicines?” Almarasson, O., et. al., The Royal Societyof Chemistry, 1889-1896, 2004. Examples of co-crystals includep-toluenesulfonic acid and benzenesulfonic acid.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds accordingto the invention can be present in different polymorphic forms, with itbeing possible for particular modifications to be metastable. Unlessstated to the contrary, the invention includes all such possiblepolymorphic forms.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc, 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions and it is understood that there are avariety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. PHARMACEUTICAL COMPOSITIONS

In one aspect, the invention relates to pharmaceutical compositionscomprising a compound represented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; wherein R³ is selectedfrom H and C1-C4 alkyl, or R² and R³ are covalently bonded and, togetherwith the intermediate atoms, comprise an optionally substituted fusedsix-membered aryl or heteroaryl ring, and a pharmaceutically acceptablecarrier or diluent.

In certain aspects, the disclosed pharmaceutical compositions comprisethe disclosed compounds and pharmaceutically acceptable salt(s) thereofas an active ingredient, a pharmaceutically acceptable carrier, and,optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, topical, andparenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically acceptable non-toxic bases oracids. When the compound of the present invention is acidic, itscorresponding salt can be conveniently prepared from pharmaceuticallyacceptable non-toxic bases, including inorganic bases and organic bases.Salts derived from such Inorganic bases include aluminum, ammonium,calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium,manganese (-ic and -ous), potassium, sodium, zinc and the like salts.Particularly preferred are the ammonium, calcium, magnesium, potassiumand sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed Include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids,”includes inorganic acids, organic acids, and salts prepared therefrom,for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof of this invention can be combined as the activeingredient in intimate admixture with a pharmaceutical carrier accordingto conventional pharmaceutical compounding techniques. The carrier cantake a wide variety of forms depending on the form of preparationdesired for administration, e.g., oral or parenteral (includingintravenous). Thus, the pharmaceutical compositions of the presentinvention can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, the compounds of theinvention, and/or pharmaceutically acceptable salt(s) thereof can alsobe administered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or morenecessary ingredients. In general, the compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both. The product can thenbe conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt of the compounds of the invention. The compounds of theinvention, or pharmaceutically acceptable salts thereof; can also beincluded in pharmaceutical compositions in combination with one or moreother therapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules are the preferredoral dosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques

A tablet containing the composition of this invention can be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets can be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention comprise acompound of the invention (or pharmaceutically acceptable salts thereof)as an active ingredient, a pharmaceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions of the present invention suitable forparenteral administration can be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage; thus, preferably should be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol and liquid polyethyleneglycol), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical use such as, for example, an aerosol, cream,ointment, lotion, dusting powder, mouth washes, gargles, and the like.Further, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing acompound of the invention, or pharmaceutically acceptable salts thereofvia conventional processing methods. As an example, a cream or ointmentis prepared by mixing hydrophilic material and water, together withabout 5 wt % to about 10 wt % of the compound, to produce a cream orointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid. It ispreferable that the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories can be conveniently formed by first admixing thecomposition with the softened or melted carrier(s) followed by chillingand shaping in moulds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining a compound of the invention, and/or pharmaceuticallyacceptable salts thereof, can also be prepared in powder or liquidconcentrate form.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

In a further aspect, the disclosed compositions are for oraladministration.

In a further aspect, the disclosed compositions further comprise atherapeutic agent that can be used to treat a retrovirus. In a stillfurther aspect, the therapeutic agent can be used to treat a retrovirusselected from the group comprising HIV-1, HIV-2, SIV, XMRV, HTLV-1,HTLV-2, HTLV-3, or HTLV-4. In yet a further aspect, the therapeuticagent can be used to treat HIV-1. In an even further aspect, thetherapeutic agent is selected from entry inhibitors, nucleoside reversetranscriptase inhibitors (NRTIs), nucleotide reverse transcriptaseinhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibitors(NNRTIs), protease inhibitors, integrase inhibitors, or maturationinhibitors, or a mixture thereof. In a still further aspect, thetherapeutic agent is selected from nucleoside reverse transcriptaseinhibitors (NRTIs), nucleotide reverse transcriptase inhibitors(NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs),protease inhibitors, or integrase inhibitors, or a mixture thereof.

C. METHODS OF USING THE COMPOUNDS AND COMPOSITIONS

The compounds disclosed herein are useful for treating or detecting aretrovirus, and more particularly for treating or detecting HIV-1. Thus,provided is a method of treating or detecting a retrovirus in a subjectcomprising the step of administering to the subject at least onecompound represented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring; and/or at least onedisclosed pharmaceutical composition in an effective dosage and amount.

The pharmaceutical compositions and methods of the present invention canfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of retroviruses, for exampleHIV-1, HIV-2, SIV, XMRV, HTLV-1, HTLV-2, HTLV-3, or HTLV-4.

In various aspects, the disclosed treatment methods can be applied to asubject, for example, a patient. In further aspects, the subject is amammal, for example, a human.

1. Treatment Methods

In one aspect, the invention relates to methods for the treatment of apatient diagnosed with a retrovirus, the method comprising the step ofadministering to the patient, together in an effective amount, acompound represented by a formula:

wherein 0 is 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl,C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring, and at least one agent knownto treat a retrovirus.

a. Activation of a Latent Retrovirus

In one aspect, the invention relates to methods of activating a latentretrovirus, and more specifically HIV-1, in a subject, the methodcomprising the step of administering to the subject an effective amountof a compound represented by a formula:

wherein n is 0 or 1; wherein R is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring.

The use of antiretroviral therapy in human immunodeficiency virus type 1(HIV-1) infected patients does not lead to virus eradication. This isdue, to a significant degree, to the fact that HIV-1 can establish ahighly stable reservoir of latently infected cells. The principlereservoir of HIV-1 latency is thought to reside in resting, CD4+ memoryT cells, which harbor integrated HIV-1 (Finzi et al. 1997). The lowfrequency of latently infected cells (1 in 10⁶ resting CD4+ T cells(Chun et al. 1997)), for which known phenotypic markers are notavailable, poses a great challenge to the study of latency in vive.

Previous studies on HIV-1 latency were based on the generation ofchronically infected cell lines, such as the ACH2 (Folks at al. 1989),JΔK (Antoni at al. 1994), and J-Lat (Jordan et al. 2003) T-cell lines,and the U1 promonocytic cell line (Folks at al. 1987). In these systems,latency was defined as a state in which integrated proviruses failed todrive efficient gene expression. However, these systems do notnecessarily reflect the latency state in vivo because the lack of viralgene expression is due to mutations in that (ACH2 and U1 (Folks at al.1989, Folks at al. 1987)) or mutations in the LTR (JΔK T-cell line(Antoni at al. 1994)). While these latency models recapitulate aplethora of mechanisms that can underlie viral latency, the focus ofthis study was in developing a more general model that did not rely onclonal probiral integration sites, and which utilized non-transformed,primary human T-cells.

A model using human fetal liver tissue in SCID-hu mice has alsogenerated a great deal of interest in the field of HIV-1 latency (Brooksat al. 2001). This model relies upon infection of thymocytes and thevast majority of latently infected cells in this system are mature,quiescent CD4+ single positive naïve T cells. This is in contrast withfindings in HIV-1 patients, where the majority of latently infectedcells are CD4+ memory T cells (Finzi at al. 1997). Although naïve andmemory cells share the characteristic of being quiescent, a likelyrequirement for HIV-1 latency in T cells (Finzi at al. 1997), there areimportant differences between these cell types that impact latency andreactivation.

More recently, a model using human, primary cells was disclosed in whichrelevant signaling pathways involved in viral reactivation are dissectedfrom latently infected memory CD4+ cells (Planelles et al. 2010).Replacement of the nef gene with one encoding the green fluorescentprotein (GFP) and using GFP as a readout allows for this primary cellmodel for the study of HIV-1 latency to be suitable for high throughputscreening (HTS) of compounds that reactivate HIV-1 from its latencystate. Disclosed herein are methods of activating a latent retrovirus,and more particularly latent HIV-1, using compounds identified in thisscreen.

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula*

wherein n is 0 or 1; wherein R¹ is selected from H,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,and N-((dimethylamino)methylene)-N-methylmethanaminiumhexafluorophosphate (V); wherein R² is selected from H and C1-C4 alkyl;and wherein R³ is selected from H and C1-C4 alkyl, or R² and R³ arecovalently bonded and, together with the intermediate atoms, comprise anoptionally substituted fused six-membered aryl or heteroaryl ring.

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a structure depicted in Table 1.

TABLE 1 Compound Name Structure 1-hydroxybenzotriazole

6-chloro-1H-benzo[d][1,2,3]triazol-1-ol

1-hydroxy-7-azabenzotriazole

4,5-dichloro-1-(cyclopentyloxy)-1H-1,2,3- benzotriazole

1-(benzoyloxy)-1H-1,2,3-benzotriazole

9-fluorenylmethyl 1-benzotriazolyl carbonate

1-{[(dimethylamino)carbonyl]oxy}-6-nitro- 1H-1,2,3-benzotriazole

1-{[(dimethylamino)sulfonyl]oxy}-6-methyl- 1H-1,2,3-benzotriazole

1-{[(dimethylamino)sulfonyl]oxy}-5-methyl- 1H-1,2,3-benzotriazole

O-(benzotriazole-1-yl)-N,N,N′,N′- tetramethyluronium tetrafluoroborate

O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′- tetramethyluroniumtetrafluoroborate

(benzotriazole-1- yloxy)dipiperidineocarbenium hexafluorophosphate

O-(benzotriazole-1-yl)-N,N,N′,N′- tetramethyluronium hexafluorophosphate

O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′- hexafluorophosphate

(benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate

O-(benzotriazole-1-yl-N,N,N′,N′- bis(pentamethylene)uraniumhexafluorophosphate

(benzotriazole-1- yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate

(benzotriazole-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate

(6-chlorobenzotriazol-1- yloxy)tripyrrolidiniophosphoniumhexafluorophosphate

(7-azabenzotriazol-1- yloxy)tripyrrolidinophosphoniumhexafluorophosphate

3-hydroxybenzo[d][1,2,3]triazin-4-(3H)-one

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² and R³ are covalently bonded and, together with theintermediate atoms, comprise an optionally substituted fusedsix-membered aryl or heteroaryl ring. In a still further aspect, R² andR³ are covalently bonded and, together with the intermediate atoms,comprise an optionally substituted fused six-membered aryl ring. In yeta further aspect, R² and R³ are covalently bonded and, together with theintermediate atoms, comprise an optionally substituted fused phenylring. In an even further aspect, R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered heteroaryl ring. In a still further aspect, R² and R³are covalently bonded and, together with the intermediate atoms,comprise an optionally substituted fused pyridine ring.

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein n is 0, R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring. In a still further aspect, nis 1.

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein R is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorphosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);and wherein each of R⁴, R⁵, and R⁶ is independently selected from H, Cl,CH₃, and NO₂.

In a still further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

wherein R¹ is selected from H andN-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V); and wherein each of R⁴, R⁵, and R⁶ is independently selected fromH, Cl, CH₃, and NO₂.

In yet a further aspect, the invention relates to a method of activatinga latent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein each of R⁴, R⁵, and R⁶ is independently selected from H, Cl,CH₃, and NO₂.

In an even further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

In a still further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein each of R⁴, R⁵, and R⁶ is independently selected from H, Cl,CH₃, or NO₂.

In a still further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V).

In a still further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

wherein R¹ is selected from H andN-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V).

In yet a further aspect, the invention relates to a method of activatinga latent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein R is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);and wherein each of R⁷, R⁸, and R⁹ is independently selected from H, Cl,CH₃, and NO₂.

In a still further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

wherein R¹ is selected from H andN-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V); and wherein each of R⁷, R⁸, and R⁹ is independently selected fromH, Cl, CH₃, and NO₂.

In yet a further aspect, the Invention relates to a method of activatinga latent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein each of R⁷, R⁸, and R⁹ is independently selected from H, Cl,CH₃, and NO₂.

In an even further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

In a further aspect, the invention relates to a method of activating alatent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar,C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V).

In a still further aspect, the invention relates to a method ofactivating a latent retrovirus in a subject, the method comprising thestep of administering to the subject an effective amount of a compoundrepresented by a formula:

wherein R¹ is selected from H andN-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V).

In yet a further aspect, the invention relates to a method of activatinga latent retrovirus in a subject, the method comprising the step ofadministering to the subject an effective amount of a compoundrepresented by a formula:

In a further aspect, the subject is a population of cells. In a stillfurther aspect, the subject is a population of cells latently infectedwith a retrovirus. In yet a further aspect, the subject is a populationof cells latently infected with HIV-1.

In a further aspect, the subject is a mammal. In a still further aspect,the subject is a human. In yet a further aspect, the subject is apatient. In an even further aspect, the subject is a patient who hasbeen diagnosed with a need for treatment of a retrovirus prior to theadministering step. In a still further aspect, the subject is a patientwho has been diagnosed with a need for treatment of HIV prior to theadministering step.

b. Agent Known to Treat a Retrovirus

In one aspect, the invention relates to methods of activating a latentretrovirus in a subject, the method further comprising treating thepatient with at least one agent that can be used to treat a retrovirus.

In various aspects, the invention relates to methods of activatinglatent HIV-1 in a subject, the method further comprising treating thepatient with at least one agent that can be used to treat HIV-1. In afurther aspect, the at least one agent is selected from entryinhibitors, nucleoside reverse transcriptase inhibitors (NRTIs),nucleotide reverse transcriptase inhibitors (NtRTIs), non-nucleosidereverse transcriptase inhibitors (NNRTIs), protease inhibitors,integrase inhibitors, or maturation inhibitors, or a mixture thereof. Ina further aspect, the at least one agent is selected from nucleosidereverse transcriptase inhibitors (NRTIs), nucleotide reversetranscriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptaseinhibitors (NNRTIs), protease inhibitors, or integrase inhibitors, amixture thereof. In yet a further aspect, the at least one agent isselected from Maraviroc, Enfuvirtide, Zidovudine, Didanosine,Zalcitabine, Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir,Tenofovir, Adefovir, Efavirenz, Nevirapine, Delavirdine, Rilpivirine,Raltegravir, Saquinavir, Ritonavir, Indinavir, Nelfinavir, orAmprenavir, or a mixture thereof. In an even further aspect, the atleast one agent is HAART.

Typically, an agent is administered in an effective amount, per itsnormal dosing instructions. In one aspect, the effective amount is atherapeutically effective amount.

2. Manufacture of a Medicament

In one aspect, the invention relates to methods for the manufacture of amedicament for the treatment of a retrovirus in a subject, the methodcomprising the step of combining an effective amount of a compoundrepresented by a formula:

wherein n is 0 or 1, wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylen)pyrrolidin-1-ium hexafluorophosphate (V), orL-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; wherein R³ is selectedfrom H and C1-C4 alkyl, or R² and R³ are covalently bonded and, togetherwith the intermediate atoms, comprise an optionally substituted fusedsix-membered aryl or heteroaryl ring, alone or in combination withanother agent, with a pharmaceutically acceptable carrier or diluent.

In a further aspect, the subject is a population of cells. In a stillfurther aspect, the subject is a population of cells latently infectedwith a retrovirus. In yet a further aspect, the subject is a populationof cells latently infected with HIV-1.

In a further aspect, the subject is a mammal. In a still further aspect,the subject is a human. In yet a further aspect, the subject is apatient. In an even further aspect, the subject is a patient who hasbeen diagnosed with a need for treatment of a retrovirus prior to theadministering step. In a still further aspect, the subject is a patientwho has been diagnosed with a need for treatment of HIV-1 prior to theadministering step.

In a further aspect, the invention further comprises treating thesubject with at least one agent that can be used to treat a retrovirus.In a still further aspect, the invention further comprises treating thesubject with at least one agent that can be used to treat HIV-1. In yeta further aspect, the at least one agent is selected from entryinhibitors, nucleoside reverse transcriptase inhibitors (NRTIs),nucleotide reverse transcriptase inhibitors (NtRTIs), non-nucleosidereverse transcriptase inhibitors (NNRTIs), protease inhibitors,integrase inhibitors, or maturation inhibitors, or a mixture thereof. Inan even further aspect, the at least one agent is selected fromnucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reversetranscriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptaseinhibitors (NNRTIs), protcase inhibitors, or integrase inhibitors, or amixture thereof. In a still further aspect, the at least one agent isselected from Maraviroc, Enfuvirtide, Zidovudine, Didanosine,Zalcitabine, Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir,Tenofovir, Adefovir, Efavirenz, Nevirapine, Delavirdine, Rilpivirine,Raltegravir, Saquinavir, Ritonavir, Indinavir, Nelfinavir, orAmprenavir, or a mixture thereof. In yet a further aspect, the at leastone agent is HAART.

3. Use of Compounds and Compositions

Also provided are the uses of compounds represented by a formula:

wherein n is 0 or 1, wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), or1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; wherein R³ is selectedfrom H and C1-C4 alkyl, or R² and R³ are covalently bonded and, togetherwith the intermediate atoms, comprise an optionally substituted fusedsix-membered aryl or heteroaryl ring, and compositions comprising thesame.

In various aspects, the use relates to the activation of a latentretrovirus in a subject. In a further aspect, the use relates to theactivation of latent HIV-1 in a subject. In a still further aspect, thesubject is a population of cells. In yet a further aspect, the subjectis a population of cells latently infected with a retrovirus. In an evenfurther aspect, the subject is a population of cells latently infectedwith HIV-1. In a still further aspect, the subject is a mammal. In astill further aspect, the subject is a human. In yet a further aspect,the subject is a patient.

4. Kits

In one aspect, the invention relates to a kit comprising a compoundrepresented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C-C6aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring and one or more of:

(a) at least one agent that can be used to treat a retrovirus;

(b) instructions for detecting a retrovirus; and

(c) instructions for treating a retrovirus.

In a further aspect, the at least one compound and the at least oneagent are co-formulated. In a further aspect, the at least one compoundand the at least one agent are co-packaged.

In a further aspect, the retrovirus is selected from a group comprisingHIV-1, HIV-2, SIV, XMRV, HTLV-1, HTLV-2, HTLV-3, or HTLV-4. In a stillfurther aspect, the retrovirus is HIV-1.

The kits can also comprise compounds and/or products co-packaged,co-formulated, and/or co-delivered with other components. For example, adrug manufacturer, a drug reseller, a physician, a compounding shop, ora pharmacist can provide a kit comprising a disclosed compound and/orproduct and another component for delivery to a patient.

It is contemplated that the disclosed kits can be used in connectionwith the disclosed methods of making, the disclosed methods of using,and/or the disclosed compositions.

5. Non-Medical Uses

Also provided are the uses of compounds represented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl,N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate,N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate(V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V),tris(dimethylamino)phosphonium hexafluorophosphate (V),1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V),and L-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V);wherein R² is selected from H and C1-C4 alkyl; and wherein R³ isselected from H and C1-C4 alkyl, or R² and R³ are covalently bonded and,together with the intermediate atoms, comprise an optionally substitutedfused six-membered aryl or heteroaryl ring as pharmacological tools inthe development and standardization of in vitro and in vivo test systemsfor the evaluation of the effects of anti-retroviral latency drugs, andmore particularly anti HIV-1 latency drugs, in laboratory animals suchas cats, dogs, rabbits, monkeys, rats and mice, as part of the searchfor new therapeutic agents that activate latent retroviruses, and morespecifically that activate latent HIV-1.

D. REFERENCES

-   Antoni B, Rabson A B, Kinter, A, Bodkin M, Poli G (1994) NF-kappa    B-dependent and -independent pathways of HIV activation in a    chronically infected T cell line. Virology 202:684-694.-   Bosque A, Planelles V (2009) Induction of HIV-1 latency and    reactivation in primary memory CD4+ T cells. Blood 113:58-65.-   Bosque A, Planelles V (2010) “Studies of HIV-1 latency in an ex vivo    model that uses primary central memory T cells”. Methods.-   Bosque A, Famiglietti M, Weyrich A S, Goulston C, Planelles V (2011)    Homeostatic Proliferation Fails to Efficiently Reactivate HIV-1    Latently Infected Central Memory CD4+ T Cells. PLOS Pathog. 7(10).-   Brooks D G, Kitchen S G, Kitchen C M, Scripture-Adams D D, Zack J    A (2001) Generation of HIV latency during thymopoiesis. Nat Med    7:459-464.-   Chun T W, Carruth L, Finzi D, et al. (1997) Quantification of latent    tissue reservoirs and total body viral load in HIV-1 infection.    Nature 387:183-188.-   Finzi D, Hermankova M, Pierson T, et al. (1997) Identification of a    reservoir for HIV-1 in patients on highly active antiretroviral    therapy. Science 278:1295-1300.-   Folks T M, Clouse K A, Justement J, et al. (1989) Tumor necrosis    factor alpha Induces expression of human immunodeficiency virus in a    chronically infected T-cell clone. Proc Natl Acad Sci USA    86:2365-2368.-   Folks T M, Justement J, Kinter A, Dinarello C A, Fauci A S (1987)    Cytokine-induced expression of HIV-1 in a chronically infected    promonocyte cell line. Science 238:800-802.-   Jordan A, Bisgrove D, Verdin E (2003) HIV reproducibly establishes a    latent infection after acute infection of T cells in vitro. Embo J    22:1868-1877.-   Planelles V, Bosque A, Methods and compositions relating to viral    latency, U.S. Patent Application 2010/0291067 A1, November 2010.

E. EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. General Methods

A novel primary cell model for the study of HIV-1 latency was adapted soas to be suitable for high-throughput screening (HTS) of new compoundsthat may reactivate HIV-1 from its latency state (Bosque and Planelles,2009; Bosque and Planelles, 2010; and patent application: Methods andcompositions relating to viral latency, Planelles V, Bosque A, U.S.Patent Application 2010/0291067 A1, November 2010).

To facilitate HTS, the nef gene was replaced with that encoding thegreen fluorescent protein (GFP). It was confirmed that deletion of nefhas no effect in latency/reactivation (Bosque and Planelles, 2009) andthat using GFP provides a faster, more economical, and equallysensitive, as compared to intracellular p24 detection, readout.

The reactivation experiments were modified to be performed in 96-wellplates, utilizing 100,000 cells per well. After reactivation, flowcytometric analysis was performed with Becton Dickinson FACSCanto IIwith a High Throughput Sampler (HTS) for microtiter plates that isavailable through the Flow Cytometry Core Facility at the University ofUtah. It takes about 30 minutes to analyze a plate, when acquiring10,000 cells per well. Analysis for each well includes (a) greenfluorescence as a direct indicator of viral reactivation; and (b)forward and side scatter as general measures of cell morphology, whichis a gross indication of viability.

2. Activation of Latent HIV-1 in Latently Infected Cultured T_(CM)

Latently infected cells were generated using healthy, uninfected DONOR78 cells that were infected with DHIV virus. Reactivation was monitoredby analysis of GFP by Flow Cytometry 72 hours after stimulation. Beadscoated with anti-CD3/anti CD28 (CD3/CD28) were used as a positivereactivation stimulus. Reactivation was done in a 96-well plate roundbottom. Screening of the Ireland Natural Product Collection, a set ofnatural products isolated from marine invertebrate animals andmicroorganisms, resulted in a positive hit in Plate 2 well C7 (FIGS. 1Aand 1B). This compound is an intermediate en route to synthesis of acyclic peptide isolated from a tunicate.

HPLC was used to generate 20 fractions based on elution time using agradient from 10-100% acetonitrile in 0.1% formic acid using aPhenomenex Kinetic C18 50×2.10 mm column (2.6 μM, 100 A). The ability ofthese fractions to activate HIV-1 was tested and compared in parallelwith the original hit. FIG. 1C shows the % of p24 positive cells in theY-axis and the % of reactivation compared to the positive control (beadscoated with anti-CD3/anti-CD28) at the top of each bar. This datareveals that the active compound is enriched in fractions 2 and 3.Further fractionation of C7 was performed and each fraction tested atthe concentration indicated in the graph (FIG. 1D). The active componentis found in fraction MS. This fraction was found to be enriched in1-hydroxybenzotriazol (HOBt) (FIG. 1E). The ability of HOBt to activatelatent HIV-1 in a dose-dependent manner compared to C7 was alsoestablished (FIG. 1F). Finally, HOBt was tested at differentconcentrations and analyzed on either Day 3, Day 5, or Day 7. Whencompared with anti-CD3/CD28, a non-druggable treatment due to its sideeffects, HOBt was found to reactive HIV-1 latency with slower kinetics(FIG. 1G).

3. Activation of Latent HIV-1 by Additional Analogs

The ability of additional triazole analogs including benzotriazole(HBt), 1-hydroxy-7-amino benzotriazole (HOAt), triazole (Ht) and1-aminobenzotriazole (ABt) to activate latent HIV-1 was also tested(Table 2). Latently infected cells were generated using healthy,uninfected DONOR 78 cells that were infected with DHIV virus.Reactivation was monitored by analysis of p24Gag by Flow Cytometry 72hours after stimulation. It was demonstrated that both HOBt and HOAtwere able to reactivate HIV-1 latency (FIGS. 2A and 2C). The activity ofboth analogs is dose dependent (FIG. 2C).

TABLE 2 Compound Structure HOBt

HOAt

6C1-HOBt

HBt

ABt

Triazole

HO-DhBt

HATU

Pyridozine

A panel of 7 derivatives of the original hit, HOBt, were tested fortheir ability to activate latent HIV-1 (FIG. 2D). HOAt is a more potentanalog than the original hit. Two additional compounds, HATU andHO-DhBt, were also able to induce viral reactivation in a dose dependentmanner and with increased efficiency.

4. Synergistic Effect of Several r-Cytokines

Latently infected cells were generated using healthy, uninfected DONOR78 cells that were infected with DHIV virus. Reactivation was monitoredby analysis of p24Gag by Flow Cytometry 72 hours after stimulation. HOBtwas tested in the presence or absence of IL-2, a γc-cytokine requiredfor survival of the cells in vivo. The ability of HOBt and HOAt toactivate latent HIV-1 in the absence of IL-2 or in the presence ofdifferent γc-cytokines, e.g. IL-4, IL-7, IL-15, IL-9, and IL-21, wastested (FIG. 3). In the absence of γc-cytokines both analogs have theability to significantly reactivate HIV-1 latency. When compared withincubation of the cells in the absence of cytokines, IL-2 and IL-7 trendtowards increasing viral reactivation. HOBt and HOAt dramaticallyincrease viral reactivation in the presence of all, IL-2, IL-4, andIL-15. IL-4 was not able to induce significant viral reactivation byitself; however, combination of IL-4 with either HOBt or HOAt inducedsignificant viral reactivation. Finally, IL-9 and IL-21 did not have aneffect in viral reactivation wither alone or in combination with HOBtand HOAt.

5. Activation of Latent HIV-1 in a STAT5 Dependent Manner

The ability of chemical inhibitors of known signaling pathways to blockviral reactivation mediated by HOAt was examined (FIG. 4A). Blockingeither NFAT or NF-κB with either Cyclosporine A (CsA) or BMS345541 didnot have a significant effect on viral reactivation induced by HOAt. CsAis able to block viral reactivation induced by αCD3/αCD28. The JAKinhibitor DBI strongly blocks viral reactivation mediated by HOAt butnot by αCD3/αCD28. Activation of JAK kinases lead to the activation ofthe transcription factors STATs. Specific inhibitors of STAT3 and STAT5were thus also studied. The STAT3 inhibitor peptide used did not have asignificant effect in viral reactivation compared with an inactivecontrol peptide. In the other hand, the STAT5 inhibitor completelyabrogates viral reactivation induced by HOAt.

The levels of STAT5 phosphorylation were analyzed by flow cytometryafter incubating cultured T_(CM) with HOAt, IL-2, or a combination ofboth, during a 24 h period (FIG. 4B). STAT5 phosphorylation could beonly slightly detected in the absence of either IL-2 or HOAt at 30 min.HOAt alone is able to induce an increase in STAT5 phosphorylation overuntreated cells. Cells incubated with IL-2 alone dramatically increasethe levels of STAT5 phosphorylation at 30 min, but these levels decreaseto basal levels after 24 hrs of incubation. Incubation of culturedT_(CM) with a combination of IL-2 and HOAt induce a drastic increase inSTAT5 phosphorylation. Moreover, the levels of STAT5 phosphorylation donot significantly decrease after 24 hrs of incubation with IL-2 andHOAt.

6. Effect on the Nuclear Localization of STAT5

The levels of nuclear localization of STAT5 after incubation of thecells with IL-2 alone or in the presence of HOAt were analyzed. Nucleartranslocation of STAT5 is required for its transcriptional activity.Both pSTAT5 and STAT5 levels increased in the nucleus of cells treatedwith HOAt at the time points indicated (FIG. 5A). Histone H3 and alphaTubulin were used as controls for purity of the fractionation.

Engagement of γc-cytokines to its receptors leads to a signaling cascadeinitiated by the phosphorylation and activation of the janus kinasesJAK1 and JAK3. These kinases then phosphorylate and activate thetranscription factors STATs. IL-2 alone or in the presence of HOAtincreases phosphorylation of JAK1 and JAK3 at similar levels (FIG. 5B).This indicates that HOAt alters STAT5 phosphorylation without alteringthe phosphorylation pattern of the JAK kinases.

STAT5 is a transcriptional activator of multiple genes. To addresswhether HOAt will increase the transcription of any STAT5 target genes,the expression levels of SOCS3, a target gene for STAT5, were analyzed.Incubation of cells with IL-2 and HOAt was found to increase the levelsof SOCS3 at 24 h post-treatment (FIG. 5C).

7. Analysis of T Cell Activation

Memory cells were generated from 3 different donors and treated witheither HOBt or anti-CD3/CD28 to analyze the effect of HOBt on T cellactivation (FIG. 6A). Analysis of T cell activation was done byanalyzing the upregulation of the activation markers CD25 and CD69 threedays post activation. HOBt does not induce general T cell activation.Analysis of T cell differentiation was done by analyzing the levels ofCCR7 and CD27 three days post activation (FIG. 6B). This revealed thatHOBt does not induce T cell differentiation measured as loss of CCR7 andCD27 expression.

In order to determine whether HOAt is inducing T cell proliferation,culter T_(CM) were incubated with HOAt in a dose dependent manner. HOAtonly induces some degree of cell proliferation at the higherconcentration tested (FIG. 6C). It has been shown previously that bothcultured T_(CM) and ex vivo T₀c express low levels of CD25 (Bosque etal. 2011). This level is increased by 37 times after TCR engagement withαCD3/αCD28, measured as mean intensity of fluorescence (values betweenparenthesis, FIG. 6D). HOAt only induced an increase of 1.4 times at themaximum concentration used.

CD69 upregulation is concomitant with T cell activation. HOAt slightlyincreases the levels of T cell activation when compared with αCD3/αCD28(FIG. 6E). These results indicate that HOAt can reactivate latent HIV-1in the absence of cellular proliferation of cellular activation.

8. Vial Reactivation Mediated by Other Stimuli

To test whether HOAt can enhance viral reactivation mediated by otherstimuli, cells were reactivated with three different agonists: PMA, aPKC agonist that has been shown to reactivate latent HIV in a NF-κBdependent manner, ionomycine, a ionophore that increases theintracellular levels of Ca²⁺ and induces the activation of thetranscription factor NFAT; and SAHA, a histone deacetylase inhibitorthat has been shown to reactive latent HIV-1 In vitro and ax vivo (FIG.7). HOAt is able to increase viral reactivation mediated by these threestimuli. These results indicate that HOAt and its derivatives can beused to synergize the activity of other anti-latency drugs.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A method of activating a latent retrovirus in a subject, the method comprising the step of administering to the subject an effective amount of a compound represented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl, N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate, N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V), tris(dimethylamino)phosphonium hexafluorophosphate (V), 1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V); wherein R² is selected from H and C1-C4 alkyl; and wherein R³ is selected from H and C1-C4 alkyl; or wherein R² and R³ are covalently bonded and, together with the intermediate atoms, comprise an optionally substituted fused six-membered aryl or heteroaryl ring. 2-11. (canceled)
 12. The method of claim 1, wherein the compound is represented by a formula:

wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl, N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate, N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V), tris(dimethylamino)phosphonium hexafluorophosphate (V), 1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V); and wherein each of R⁴, R⁵, and R⁶ is independently selected from H, Cl, CH₃, and NO₂.
 13. The method of claim 1, wherein the compound is represented by a formula:

wherein R¹ is selected from H and N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V); and wherein each of R⁴, R⁵, and R⁶ is independently selected from H, Cl, CH₃, and NO₂.
 14. The method of claim 1, wherein the compound is represented by a formula:

wherein each of R⁴, R⁵, and R⁶ is independently selected from H, Cl, CH₃, and NO₂.
 15. The method of claim 1, wherein the compound is represented by a formula:


16. The method of claim 1, wherein the compound is represented by a formula:


17. The method of claim 1, wherein the compound is represented by a formula:

wherein each of R⁴, R⁵, and R⁶ is independently selected from H, Cl, CH₃, or NO₂.
 18. The method of claim 1, wherein the compound is represented by a formula:


19. The method of claim 1, wherein the compound is represented by a formula:

wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl, N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate, N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V), tris(dimethylamino)phosphonium hexafluorophosphate (V), 1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V). 20-21. (canceled)
 22. The method of claim 1, wherein the compound is represented by a formula:

wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl, N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate, N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V), tris(dimethylamino)phosphonium hexafluorophosphate (V), 1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V); and wherein each of R⁷, R⁸, and R⁹ is independently selected from H, Cl, CH₃, and NO₂.
 23. The method of claim 1, wherein the compound is represented by a formula:

wherein R¹ is selected from H and N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V); and wherein each of R⁷, R⁸, and R⁹ is independently selected from H, Cl, CH₃, and NO₂.
 24. (canceled)
 25. The method of claim 1, wherein the compound is represented by a formula:


26. The method of claim 1, wherein the compound is represented by a formula:

wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar, C(O)N(CH₃)₂, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl, N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate, N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V), tris(dimethylamino)phosphonium hexafluorophosphate (V), 1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V). 27-41. (canceled)
 42. The method of claim 1, further comprising treating the patient with at least one agent that can be used to treat HIV-1.
 43. The method of claim 42, wherein the at least one agent is selected from entry inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, integrase inhibitors, or maturation inhibitors, or a mixture thereof.
 44. The method of claim 42, wherein the at least one agent is selected from nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, or integrase inhibitors, or a mixture thereof.
 45. The method of claim 42, wherein the at least one agent is selected from Maraviroc, Enfuvirtide, Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir, Tenofovir, Adefovir, Efavirenz, Nevirapine, Delavirdine, Rilpivirine, Raltegravir, Saquinavir, Ritonavir, Indinavir, Nelfinavir, or Amprenavir, or a mixture thereof.
 46. The method of claim 42, wherein the at least one agent is HAART.
 68. A pharmaceutical composition comprising a compound represented by a formula:

wherein n is 0 or 1; wherein R¹ is selected from H, C1-C4 alkyl, C1-C6 aryl, C(O)Ar, SO₂N(CH₃)₂, fluorenylmethyloxycarbonyl, N-((dimethylamino)methylene)-N-methylmethanaminium tetrafluoroborate, N-((dimethylamino)methylene)-N-methylmethanaminium hexafluorophosphate (V), tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V), tris(dimethylamino)phosphonium hexafluorophosphate (V), 1-(pyrrolidin-1-ylmethylene)pyrrolidin-1-ium hexafluorophosphate (V), and 1-(piperidin-1-ylmethylene)piperidin-1-ium hexafluorophosphate (V); wherein R² is selected from H and C1-C4 alkyl; wherein R³ is selected from H and C1-C4 alkyl; or wherein R² and R³ are covalently bonded and, together with the intermediate atoms, comprise an optionally substituted fused six-membered aryl or heteroaryl ring; and a pharmaceutically acceptable carrier or diluent.
 69. (canceled)
 70. The composition of claim 68, further comprising a therapeutic agent that can be used to treat a retrovirus. 71-74. (canceled) 